Refined petroleum products have been a major energy source for many decades. Unfortunately, the prices for both refined and crude petroleum based products have risen dramatically in recent years due to excess demand and dwindling supply. Moreover, petroleum products, when burned, contribute substantially to the ongoing global warming crisis. As a result of the many issues associated with petroleum products, there is substantial interest in exploring alternative energy sources. One alternative to petroleum-based fuel is ethanol.
Ethanol is typically produced by yeast fermentation of either sucrose or glucose, the structures of which are shown in FIG. 1. (Wang, M., et al. Effect of Fuel Ethanol Use on Fuel-Cycle Energy and Greenhouse Gas Emission; Argonne National Laboratory: Argonne, 1999.). The viability of replacing petroleum-based fuels with ethanol has already been successfully demonstrated. In Brazil, for example, sugar cane-derived ethanol has largely replaced petroleum-based fuels. (Sperling, D.; Gordon, D., Two billion cars: driving toward sustainability. Oxford University Press: New York, 2009.).
Although ethanol has shown promise as an alternative fuel source, there are many obstacles that have hampered the growth of ethanol production in the United States. Specifically, the raw materials necessary to produce ethanol, e.g., sucrose and glucose, are common food stuffs. Sucrose, for example, is a non-reducing disaccharide consisting of glucose and fructose, and is produced primarily from cane sugar or sugar beets. Glucose, produced by the hydrolysis of plant starches (repeating polymers of α-linked glucosides), is derived predominantly from corn. The demand for the raw materials necessary to produce ethanol competes with the demand for food, resulting in increased prices for both food and fuel. The potential benefits of ethanol derived from corn are further diminished by studies that have shown that corn-based ethanol could have a net climate warming effect. (Crutzen, P. J., et al., Atmospheric Chemistry and Physics 2008, 8, (2), 389-395.)
In an attempt to mitigate some of the issues related to the production corn-based ethanol, the ethanol industry has turned its attention to methods of developing glucose from cellulose. Cellulose is a polymer consisting of β-1,4-linked glucosides and can be found in nearly all plant materials. Thus, an efficient method of hydrolyzing cellulose into glucose could allow ethanol production facilities to access the estimated 1011 tons per year of cellulose normally produced by plants on earth.
Large amount of cellulose could even be obtained from crop remains such as corn stover, cane bagasse, and even cellulose-based trash. These materials would be sustainable, easy to collect, and very inexpensive (Saha, B. C., et al. In Fuel ethanol production from corn fiber—Current status and technical prospects, 1998; Humana Press Inc: 1998; pp 115-125; Tucker, M. P., et al. In Conversion of distiller's grain into fuel alcohol and a higher-value animal feed by dilute-acid pretreatment, 2004; Humana Press Inc: 2004; pp 1139-1159.). Another advantage of cellulose over cane sugar is that cellulose hydrolysis will yield only glucose, which yeast favor over sucrose.
Even though cellulose is easily the most abundant biological material on earth, it is not trivial to hydrolyze cellulose directly to glucose. Given the difficulties associated with the hydrolysis, most efforts at large-scale cellulose hydrolysis have been focused on using cellulases, a class of enzymes that catalyze the hydrolysis of cellulose. Research has also focused on the preparation of modified enzymes engineered to be more stable under the extreme high temperatures and pH conditions required to hydrolyze cellulose. (Sun, Y. et al. Bioresource Technology 2002, 83, (1), 1-11; Wright, J. D. Chemical Engineering Progress 1988, 84, (8), 62-74.)
Despite some success in preparing cellulases capable of hydrolyzing cellulose to glucose, even the most heat-stable cellulases are expensive and relatively short-lived. Moreover, enzyme-based hydrolysis typically requires several days to achieve desirable reactions. It is also difficult to separate and reuse cellulase enzymes, making any process using these reagents more expensive. The combination of these various issues greatly impedes the economical production of ethanol from lignocellulosic material. It would therefore be useful to develop and use one or more inorganic catalysts that can mimic an exoglucosidase (an enzyme that cleaves a terminal glucose residue from a cellulose oligo- or polysaccharide) and/or an endoglucosidase (an enzyme that cleaves the glucose polymer at an internal linkage).
Inorganic catalysts, unlike their biological counterparts, can successfully tolerate harsh conditions and can be used repeatedly without loss of activity. Moreover, even if an inorganic catalyst were less active then presently known enzymes, the inorganic catalyst could have significant commercial importance. For example, a silica-based zeolite 100 times less active than a corresponding enzyme, but 1000 times less expensive to produce, and 100 times more stable under the conditions necessary for cellulose hydrolysis, would be a commercially attractive alternative to enzyme based technology.
Recent efforts to prepare inorganic catalysts for various purposes have focused on a strategy employing molecular imprinting. (Gupta, R., et al. Biotechnology Advances 2008, 26, (6), 533-547; Katz, A., et al. Nature 2000, 403, (6767), 286-289.) In molecular imprinting, an imprinting template acts as a form around which cross-linkable monomers are co-polymerized to form a cast-like shell. Without wishing to be bound by any particular theory, it is believed that the monomers in a given imprinting reaction form a complex with the template through covalent and/or non-covalent interactions. The monomers are subsequently polymerized in the presence of the template.
After polymerization, the imprinting template is removed, exposing cavities that are complementary to the template in size and shape. These cavities, essentially negative images of the imprinting templates, are subsequently capable of selectively rebinding the templates, or molecules similar to the templates. The template-free polymer or copolymer can be referred to as a “molecularly imprinted polymer” (“MIP”). MIPs possess the most important features of biological receptors-recognition.
MIPs can comprise cross linked polymers, as described above. They can also comprise amorphous metal oxides or zeolites. Metal oxides and zeolites can be imprinted using a variety of known techniques. In some cases, the cavities or pores produced are an induced fit for polymers of the imprinting molecules. These polymers can subsequently be hydrolyzed by the MIP using the appreciable thermal energy that the imprinted structures can withstand.
A wide variety of templates are suitable for preparing MIPs including, but not limited to, pharmaceuticals, pesticides, amino acids, peptides, nucleotide bases, steroids, and sugars. Derivatives of the target molecule can be used as a template. These derivatives typically mimic the three dimensional structure and functionality of the parent target molecule, but result in MIPs with improved properties. Examples of improved properties include, but are not limited to increased rates of catalysis, longer catalyst life, increased stability at high temperature, or increased stability at various pHs.
There are currently at least four approaches to molecular imprinting. These techniques use various molecules for imprinting, including substrate analogues, transition state analogues, product analogues, and cofactors.
The use of a substrate analogue involves the use of a compound that mimics the reaction complex between the substrate and the matrix. Catalytic groups are introduced in the site by ‘baiting’ them with the print species and will subsequently act catalytically upon binding of the true substrate. Substrate inhibition can be avoided as the bait molecule may bear little resemblance to either reaction species. An early attempt at using this strategy was the preparation of imprinted matrices with esterolytic activity. In these matrices, cobalt(II) ions were used to coordinate catalytically active vinylimidazole groups and the template during the imprinting process. Subsequent introduction of the substrates (p-nitrophenyl esters of methionine or leucine) to the sites resulted in accelerated and substrate-specific hydrolysis of activated amino acid analogues.
This strategy has also been evaluated to study the mechanism involved in the dehydrohalogenation of α-fluoroketones. In this reaction, the imprinted-matrix facilitated catalysis led to an increase in dehydrofluorination rate compared with the solution reaction by a factor of about 600 (calculated by kcat/kuncat wherein Kcat=where rate of the catalyzed reaction and kuncat is the rate of the uncatalyzed reaction). The same reaction could also be achieved by the reverse system, in which a carboxylic acid print molecule was used as bait for positioning amino groups in the polymer.
The isomerization of benzisoxazoles has also been studied. In this study, indole was used as a substrate analogue for positioning pyridinyl groups in a matrix. The resulting matrix was shown to be remarkably efficient, with a rate enhancement [(kcat/KM)/kuncat] of 40,000 over the catalyzed solution reaction. A substrate strategy has also been employed for the dinitrophenolysis of benzoic anhydrides, in which the corresponding benzamide was used as a template.
Another approach to catalysis by molecularly imprinted materials is the use of transition state analogues (TSAs) as templates. When using transition state analogues, the recognition site of the matrix is designed to stabilize the transition state of a given reaction, thereby lowering the transition energy of the reaction and leading to an enhanced reaction rate. For example, the transition state of ester hydrolysis can be mimicked by phosphonate derivatives, a feature that has been used in the preparation of several molecularly imprinted materials.
The TSA approach was followed when a phosphonate ester was imprinted using a polymerizable amidine derivative. The catalyst was tested on the hydrolysis of carboxylic acid esters analogous to the phosphonate esters, resulting in enhanced catalytic efficiencies. The relative reaction rate was 100 times higher for the imprinted matrices compared with the uncatalyzed solution reaction and five times more efficient than for a reference polymer.
Product analogues have also been used as templates in a number of cases. Catalytic polymers prepared using these templates, however, can be sensitive to inhibition. Choice of appropriate templates, though, can overcome this inhibition. For example, product analogues have been used as templates to create catalysts for a Diels-Alder reaction between tetrachlorothiophene dioxide and maleic anhydride.
The Diels-Alder reaction has an inherent entropic barrier and, for efficient catalysis to occur, sufficient stabilization of the diene and dienophile needs to be accomplished. In this study, a product analog, chlorendic anhydride, was used a template in the imprinting protocol. As a result of the judicious choice of a template, the inhibition was minimized. The resulting matrices enhanced the rate of the Diels-Alder reaction about 270-fold.
Another strategy is the use of imprinted cofactors. Natural enzymes frequently use cofactors to enable efficient catalysis because the protein(s) being acted upon are often devoid of side chains carrying reactive electrophilic groups. The cofactors enable group transfer reactions. In addition, cofactors can easily be transported to a location where they are needed. Such cofactors may involve metal ions that can act as Lewis acids in, for example, facilitating polarization of carbonyl groups and binding water molecules. In addition, coenzymes can facilitate a number of reactions, such as redox processes, (de)carboxylations and transaminations.
For example, the chemistry involved in enzymatic reactions using the coenzyme pyridoxalphosphate, common to many enzymes, was employed in an imprinting protocol. N-pyridoxyl-L-phenylalanine anilide was used as the template. The resulting imprinted polymer was analyzed for its ability to catalyze the formation of adducts between free pyridoxal and phenylalanine. An eightfold rate enhancement was recorded compared with a reference polymer.
An example of metal-coordination-assisted catalysis has been reported in the preparation of a class II aldolase mimic. A complex of an analogue of a reactive intermediate product (dibenzoylmethane) with cobalt(II) ions was imprinted in conjunction with 4-vinylpyridine in a polystyrene-based copolymer system. The polymer was capable of catalyzing the condensation of acetophenone and benzaldehyde to produce chalcone and the resulting activity was eight times higher than the solution reaction. Substrate selectivity and true turnover could be recorded.
In addition to the above discussed embodiments, studies have also demonstrated that mesoporous organic-inorganic silica catalysts can be used for hydrolysis of subunits of cellulose. See, e.g. Bootsma, J. A., et al., Applied Catalysis a-General 2007, 327, (1), 44-51 and Bootsma, J. A., et al., Bioresource Technology 2008, 99, (12), 5226-5231. The activation energy of the processes described in these references was found to be similar to those reported for cellulose hydrolysis reactions using homogeneous organic acids. Although these studies show the feasibility of using inorganic catalysts for hydrolysis of cellulose, the silica catalysts described in these references lack the specificity of natural enzymes and cause substantial degradation of cellulose subunits to compounds other than glucose.
Thus, in order to achieve mass production of ethanol from cellulose, there is a long felt, but unmet need for scalable, chemically robust, and economical processes to hydrolyze cellulose to glucose for subsequent fermentation. The present invention meets this need.